agar and Neoplasm-Metastasis

The spread of lung cancer cells to distant sites represents a common event associated with poor prognosis. A fraction of tumor cells named cancer stem cells (CSCs) have the ability to overcome therapeutic stress and remain quiescent. However, whether these CSCs have also the capacity to initiate and sustain metastasis remains unclear. Here, we used tumor sphere cultures (TSC) isolated from mouse and human lung cancer models to enrich for CSCs, and assessed their metastatic potential as compared to non-CSCs. As expected, TSC overexpressed a variety of stem cell markers and displayed chemoresistance. The CSC phenotype of TSC was confirmed by their higher growth ability in soft agar and tumorigenic potential in vivo, despite their reduced in vitro cell growth kinetics. Surprisingly, the appearance of spontaneous lung metastases was strongly delayed in mice injected with TSC as compared to non-TSC cells. Similarly, this finding was confirmed in several other models of metastasis, an effect associated with a retarded colonization activity. Interestingly, such delay correlated with a quiescent phenotype whose underlined mechanisms included an increase in p27 protein and lower phospho-ERK1/2 levels. Thus, these data suggest that cells enriched for CSC properties display an impaired metastatic activity, a finding with potential clinical implications.

Topics: Agar; Animals; Antineoplastic Agents; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Lung Neoplasms; Mice; Mice, Nude; Mice, Transgenic; Neoplasm Metastasis; Neoplastic Stem Cells; Osteolysis; p38 Mitogen-Activated Protein Kinases; Phenotype; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Spheroids, Cellular

The objective of this study was to use the established xenograft model of human melanoma (C8161.9) to test a pharmacological approach to the effect of the metastasis suppressor KISS1. A KISS1 analog was used to inhibit the metastatic development of C8161.9 cells in nude mice. Further experiments were performed to test the validity of the C8161.9 model and test the connection between KISS1 expression and loss of metastatic potential. New clones of C8161.9 cells were obtained, with or without KISS1 expression, and were tested for metastasis formation. The absence of benefit in survival with the KISS1 analog compared with PBS prompted us to revisit the C8161.9 model. We found that the cells expressing KISS1, used in the previous study and obtained by transfection and single-cell cloning, were defective for both formation of orthotopic tumors and metastases. In mixing experiments, these cells could not suppress orthotopic tumor growth of KISS1-negative C8161.9 cells, suggesting that the suppression of metastasis by C8161.9-KISS1 cells may be intrinsic to the selected clone rather than related to KISS1 expression. Isolation of clones from parental C8161.9 cells in soft agar yielded cell populations that phenotypically and genotypically mimicked the KISS1-positive clone. In addition, new clones expressing KISS1 did not show any decrease in metastatic growth. These data demonstrate the heterogeneity of cell types in the C8161.9 cell line and the high risk of artifact linked to single-cell selection. A different xenograft model will be necessary to evaluate the use of KISS1 analogs as antimetastatic therapy.

Topics: Agar; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Culture Media, Conditioned; Enzyme-Linked Immunosorbent Assay; Humans; Kisspeptins; Melanoma; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Nucleic Acid Hybridization

An approach that facilitates rapid isolation and characterization of tumor cells with enhanced metastatic potential is highly desirable. Here, we demonstrate that plating GI-101A human breast cancer cells on hard (0.9%) agar selects for the subpopulation of metastasis-initiating cells. The agar-selected cells, designated GI-AGR, were homogeneous for CD44(+) and CD133(+) and five times more invasive than the parental GI-101A cells. Moreover, mice injected with GI-AGR cells had significantly more experimental brain metastases and shorter overall survival than did mice injected with GI-101A cells. Comparative gene expression analysis revealed that GI-AGR cells were markedly distinct from the parental cells but shared an overlapping pattern of gene expression with the GI-101A subline GI-BRN, which was generated by repeated in vivo recycling of GI-101A cells in an experimental brain metastasis model. Data mining on 216 genes shared between GI-AGR and GI-BRN breast cancer cells suggested that the molecular phenotype of these cells is consistent with that of cancer stem cells and the aggressive basal subtype of breast cancer. Collectively, these results demonstrate that analysis of cell growth in a hard agar assay is a powerful tool for selecting metastasis-initiating cells in a heterogeneous population of breast cancer cells, and that such selected cells have properties similar to those of tumor cells that are selected based on their potential to form metastases in mice.

Topics: Agar; Animals; Blotting, Western; Brain Neoplasms; Breast Neoplasms; Cell Culture Techniques; Cell Proliferation; Female; Gene Expression; Gene Expression Profiling; Humans; Mice; Mice, Nude; Neoplasm Metastasis; Neoplastic Stem Cells; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured

The aim of this study was to characterize the oncogenic function and mechanism of Cathepsin Z (CTSZ) at 20q13.3, a frequently amplified region in hepatocellular carcinoma (HCC). Real-time PCR were used to compare CTSZ expression between paired HCC tumor and non-tumor specimens. CTSZ gene was stably transfected into HCC line QGY-7703 cells and its role in tumorigenicity and cell motility was characterized by soft agar, wound-healing, transwell invasion and cell adhesion assay, and tumor xenograft mouse model. Western blot analysis was used to study expression of proteins associated with epithelial-mesenchymal transition (EMT).Upregulation of CTSZ was detected in 59/137 (43%) of primary HCCs, which was significantly associated with advanced clinical stage (P = 0.000). Functional study found that CTSZ could increase colony formation in soft agar and promote cell motility. Further study found that the metastatic effect of CTSZ was associated with its role in inducing epithelial-mesenchymal transition (EMT) by upregulating mesenchymal markers (fibronectin and vimentin) and downregulating epithelial markers (E-cadherin and α-catenin). In addition, CTSZ could also upregulate proteins associated with extracellular matrix remodeling such as MMP2, MMP3 and MMP9. Taken together, our data suggested that CTSZ was a candidate oncogene within the 20q13 amplicon and it played an important role in HCC metastasis.

Topics: Agar; alpha Catenin; Animals; Cadherins; Carcinoma, Hepatocellular; Cathepsin Z; Cell Adhesion; Cell Movement; Epithelial-Mesenchymal Transition; Fibronectins; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Matrix Metalloproteinases; Mice; Mice, SCID; Neoplasm Metastasis; Neoplasm Transplantation; Vimentin; Wound Healing

The matrix glycoprotein, fibronectin, stimulates the proliferation of non-small cell lung carcinoma in vitro through α5β1 integrin receptor-mediated signals. However, the true role of fibronectin and its receptor in lung carcinogenesis in vivo remains unclear. To test this, we generated mouse Lewis lung carcinoma cells stably transfected with short hairpin RNA shRNA targeting the α5 integrin subunit. These cells were characterized and tested in proliferation, cell adhesion, migration, and soft agar colony formation assays in vitro. In addition, their growth and metastatic potential was tested in vivo in a murine model of lung cancer. We found that transfected Lewis lung carcinoma cells showed decreased expression of the α5 gene, which was associated with decreased adhesion to fibronectin and reduced cell migration, proliferation, and colony formation when compared with control cells and cells stably transfected with α2 integrin subunit in vitro. C57BL/6 mice injected with α5-silenced cells showed lower burden of implanted tumors, and a dramatic decrease in lung metastases resulting in higher survival as compared with mice injected with wild-type or α2 integrin-silenced cells. These observations reveal that recognition of host- and/or tumor-derived fibronectin via α5β1 is important for tumor growth both in vitro and in vivo, and unveil α5β1 as a potential target for the development of anti-lung cancer therapies.

Topics: Agar; Animals; Carcinoma, Lewis Lung; Cell Adhesion; Cell Movement; Cell Proliferation; Disease Progression; Fibronectins; Gene Silencing; Injections, Intravenous; Injections, Subcutaneous; Integrin alpha5beta1; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasm Transplantation; RNA, Small Interfering; Survival Analysis; Tumor Stem Cell Assay

Myopodin was previously reported as a gene that was frequently deleted in prostate cancer. This gene shares significant homology with a cell shape-regulating gene, synaptopodin. Myopodin was shown to bind actin and to induce actin bundling when cells were stimulated. To clarify the functional role of myopodin in prostate cancer, several assays were performed to evaluate the tumor suppression activity of myopodin. Our results indicate that myopodin inhibits tumor growth and invasion both in vitro and in vivo. The activity of tumor suppression of myopodin is located at the C-terminus region. To further evaluate the role of myopodin in suppressing the invasiveness of prostate cancer, an expression analysis of myopodin protein was performed in prostate tissues. The results indicate that down-regulation of myopodin expression occurs mostly in invasive stages of prostate cancer, implying a potential invasion suppression role for myopodin in prostate cancer. In addition, hemizygous deletion and down-regulation of myopodin expression occur in three aggressive prostate cancer cell lines. All these results support the hypothesis that myopodin functions as a tumor suppressor gene to limit the growth and to inhibit the metastasis of cancer cells.

Topics: Agar; Animals; Cell Division; Cell Line, Tumor; Collagen; Disease Progression; Down-Regulation; Drug Combinations; Gene Deletion; Humans; Immunoblotting; Immunohistochemistry; Laminin; Male; Mice; Mice, SCID; Microfilament Proteins; Neoplasm Metastasis; Neoplasm Transplantation; Prostatic Neoplasms; Protein Structure, Tertiary; Proteoglycans; Time Factors

Manganese superoxide dismutase (MnSOD) activity is generally lower in cancer cells when compared with their normal cell counterparts. Many studies have shown that replacing the diminished MnSOD activity leads to inhibition of the malignant phenotype. We sought to overexpress MnSOD in a chemically transformed, malignant rat cell line with low endogenous MnSOD activity to determine the effect on the malignant phenotype. After MnSOD cDNA transfection, clonal populations were characterized at the molecular level for protein, RNA, and DNA, as well as for in vitro and in vivo growth and in vivo lung metastasis. MnSOD transfectants, which both under- and overexpressed MnSOD protein, were identified. These transfectants demonstrated variations in glutathione peroxidase and catalase activity levels, indicating differences in peroxide-generating versus peroxide-metabolizing enzymes (antioxidant imbalance); these differences were suggestive of alterations in their abilities to metabolize peroxide when compared with the parental cell line. In addition, these transfectants demonstrated reductions in both in vitro and in vivo growth, as well as a reduction in metastatic potential, which correlated with antioxidant imbalance. These results suggest that the tumor suppressive effect of MnSOD overexpression is in part mediated by an antioxidant imbalance resulting in the reduced capacity to metabolize increased levels of intracellular peroxides.

Topics: Agar; Animals; Antioxidants; Blotting, Southern; Blotting, Western; Catalase; Cell Line, Transformed; Cell Line, Tumor; Chloramphenicol O-Acetyltransferase; DNA; DNA, Complementary; Glutathione Peroxidase; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Metastasis; Peroxides; Phenotype; Plasmids; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA; Superoxide Dismutase; Time Factors; Transfection

The small GTPase RhoA has been implicated in the regulation of cell morphology, motility, and transformation, but the role of RhoA protein in the carcinogenesis of gastric cancer remains unclear. In the present study, we have analyzed the expression status of the RhoA protein in human gastric cancer cells and tissues and investigated the possible involvement of RhoA in regulating the malignant phenotype of gastric cancer cells.. RhoA expression was analyzed by immunohistochemistry and Western blot in gastric cancer tissues and cell lines. The RhoA-specific small interfering RNA (siRNA) vector was designed and constructed. We examined the role of RhoA in the malignant phenotype of gastric cancer cells by using siRNA knockdown and dominant-negative RhoA mutant suppression of endogenous RhoA activity.. RhoA was found frequently overexpressed in gastric cancer tissues and cells compared with normal tissues or gastric epithelial cells. RhoA-specific siRNA could specifically and stably reduce RhoA expression up to 90% in AGS cells. Both RhoA-specific siRNA and dominant-negative RhoA expressions could significantly inhibit the proliferation and tumorigenicity of AGS cells and enhance chemosensitivity of the cancer cells to Adriamycin and 5-fluorouracil.. RhoA may play a critical role in the carcinogenesis of gastric cancer, and the interference of RhoA expression and/or activity could provide a novel avenue in reversing the malignant phenotype of gastric cancer cells.

Topics: Agar; Animals; Antibiotics, Antineoplastic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Blotting, Western; Cell Cycle; Cell Differentiation; Cell Line, Transformed; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; DNA; Dose-Response Relationship, Drug; Doxorubicin; Epithelial Cells; Flow Cytometry; Fluorouracil; Genes, Dominant; Humans; Immunohistochemistry; Mice; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Oligonucleotides; Phenotype; rhoA GTP-Binding Protein; RNA, Small Interfering; Stomach Neoplasms; Tetrazolium Salts; Thiazoles; Time Factors; Transfection; Wound Healing

The molecular mechanism leading to the cancer metastasis to bone is poorly understood but yet determines prognosis and therapy. Here, we define a new molecular pathway that may account for the extraordinarily high osteotropism of prostate cancer. By using SPARC (secreted protein, acidic and rich in cysteine)-deficient mice and recombinant SPARC, we demonstrated that SPARC selectively supports the migration of highly metastatic relative to less metastatic prostate cancer cell lines to bone. Increased migration to SPARC can be traced to the activation of integrins alphaVbeta3 and alphaVbeta5 on tumor cells. Such activation is induced by an autocrine vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)-2 loop on the tumor cells, which also supports the growth and proliferation of prostate cancer cells. A consequence of SPARC recognition by alphaVbeta5 is enhanced VEGF production. Thus, prostate cancer cells expressing VEGF/VEGFR-2 will activate alphaVbeta3 and alphaVbeta5 on their surface and use these integrins to migrate toward SPARC in bone. Within the bone environment, SPARC engagement of these integrins will stimulate growth of the tumor and further production of VEGF to support neoangiogenesis, thereby favoring the development of the metastatic tumor. Supporting this model, activated integrins were found to colocalize with VEGFR-2 in tissue samples of metastatic prostate tumors from patients.

Topics: Agar; Animals; Bone and Bones; Bone Neoplasms; Cell Division; Cell Movement; Flow Cytometry; Humans; Immunohistochemistry; Integrin alphaVbeta3; Integrins; Male; Mice; Models, Biological; Neoplasm Metastasis; Neoplasm Transplantation; Osteonectin; Prostatic Neoplasms; Receptors, Vitronectin; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured; Vascular Endothelial Growth Factor Receptor-2

Long-term elevation of metastasis suppressor gene expression in micrometastases represents a novel therapeutic strategy for breast and other cancers. We searched for well-tolerated compounds that could elevate Nm23 metastasis suppressor expression in metastatic human breast cancer cell lines.. MDA-MB-435 and MDA-MB-231 human breast carcinoma cells were treated with dexamethasone or medroxyprogesterone acetate (MPA) in cultures containing either charcoal-stripped serum or FCS. Aspects of nm23 expression and function were determined.. Previous investigation of the nm23-H1 promoter suggested that glucocorticoids may contribute to the elevation of Nm23-H1 expression. Dexamethasone elevated Nm23-H1 and Nm23-H2 protein levels in two metastatic human breast carcinoma cell lines 2-3-fold over a 4-day time course when cultured in steroid-free culture medium, with high-dose inhibition, via a traditional transcriptional mechanism. Elevation of Nm23-H1 expression was not observed using FCS-containing culture medium, which contains endogenous levels of corticosteroids, limiting the potential in vivo use of dexamethasone. MPA was investigated as a glucocorticoid receptor agonist. MPA elevated breast carcinoma Nm23-H1 protein expression 3-fold over a 10 nM to 1 micro M dose range when cultured in steroid-free or FCS-containing medium, with a shorter time course. Elevation of Nm23-H1 expression in the presence of endogenous corticosteroids found in FCS involved a distinct, glucocorticoid receptor-dependent, posttranscriptional mechanism of action. MPA had no effect on proliferation in vitro but reduced the soft agar colonization of metastatic breast cancer cell lines by approximately 50%.. MPA represents a first generation lead agent for the elevation of Nm23-H1 metastasis suppressor expression and the inhibition of metastatic colonization.

Topics: Agar; Antineoplastic Agents, Hormonal; Blotting, Western; Breast Neoplasms; Cell Division; Cell Line, Tumor; Contraceptives, Oral, Synthetic; Dexamethasone; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Glucocorticoids; Humans; Medroxyprogesterone; Medroxyprogesterone Acetate; Mutagenesis; Neoplasm Metastasis; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Promoter Regions, Genetic; Protein Biosynthesis; RNA Processing, Post-Transcriptional; Time Factors; Transcription, Genetic; Transfection

Nm23 gene family has been associated with metastasis suppression and differentiation. We studied DR-nm23 during neuroblastoma cells differentiation. DR-nm23 expression increased after retinoic acid induction of differentiation in human cell lines SK-N-SH and LAN-5.. In several cell lines, overexpression of DR-nm23 was associated with more differentiated phenotypes. SK-N-SH cells increased vimentin expression, increased deposition of collagen type IV, modulated integrin expression, and underwent growth arrest; the murine neuroblastoma cell line N1E-115 showed neurite outgrowth and a striking enhancement of beta1 integrin expression. Up-regulation of beta1 integrin was specifically responsible for the increase in the adhesion to collagen type I-coated plates. Finally, cells overexpressing DR-nm23 were unable to growth in soft agar.. In conclusion, DR-nm23 expression is directly involved in differentiation of neuroblastoma cells, and its ability to affects the adhesion to extracellular substrates and to inhibit growth in soft agar suggests an involvement in the metastatic potential of neuroblastoma.

Topics: Agar; Animals; Cell Adhesion; Cell Differentiation; Collagen; Culture Media; Gene Expression Regulation, Neoplastic; Humans; Integrin beta1; Intracellular Signaling Peptides and Proteins; Isoenzymes; Mice; Monomeric GTP-Binding Proteins; Neoplasm Metastasis; Neoplasm Proteins; Neurites; Neuroblastoma; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Phenotype; Recombinant Fusion Proteins; Transcription Factors; Transfection; Tumor Cells, Cultured; Vimentin

To search for anti-cancer agents, a screening system for Ras signal inhibitors was developed using a NIH3T3 cell line with an introduced reporter gene which is controlled by the Ras-responsive element (RRE). With this screening system, malolactomycin D was identified as a selective inhibitor of transcription from the RRE. This compound was found to preferentially inhibit the anchorage-independent growth rather than the anchorage-dependent growth of Ras-transformed NIH3T3 cells. The expression of matrix metalloproteinases MMP-1 and MMP-9, which have RRE in their promoters, were reduced by treatment with malolactomycin D at the translational and transcriptional levels. Analysis of the activity of mitogen-activated protein (MAP) kinases, which play important roles in transduction of the Ras signal, showed that malolactomycin D inhibits the activation of p38 MAP kinase and Jun N-terminal-kinase (JNK) but not extracellular signal-regulated kinase 1 or 2 (ERK1 or 2). These findings suggest that by inhibiting the pathway that leads to the activation of p38 MAP kinase and JNK, malolactomycin D suppresses the expression of MMPs. Since MMPs play important roles in metastasis and maintenance of the microenvironment of tumor cells, both of which facilitate tumor growth, the inhibition of MMPs by malolactomycin D is believed to contribute to its ability to inhibit Ras-mediated tumorigenesis.

Topics: 3T3 Cells; Agar; Animals; Anti-Bacterial Agents; Antifungal Agents; Blotting, Northern; Blotting, Western; Cell Division; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; JNK Mitogen-Activated Protein Kinases; Luciferases; Macrolides; Matrix Metalloproteinase 1; Matrix Metalloproteinase 9; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Metastasis; p38 Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; ras Proteins; Time Factors; Transcription, Genetic; Transfection

An immunohistochemical and ultrastructural study of human melanoma colonies grown in soft agar for up to 50 days was performed. Three morphological variants of developing tumor colonies are reported: 1) large light colonies, 2) small dark colonies, and 3) smooth-edged colonies. The large light colony variant is the most frequently observed in the soft agar assay (approximately 70%), followed by the dark colony variant (approximately 27%), and the smooth-edged colony variant (approximately 3%). Major morphological characteristics are associated with each variant, as shown with light microscopy (LM) and transmission electron microscopy (TEM). Both LM and TEM analyses demonstrated that the large light colony variant was hypomelanotic and contained a microfibrillar extracellular matrix (ECM). The small dark colony variant was found to be hypermelanotic and contained a less demonstrable ECM. The smooth-edged variant has an encapsulated periphery, no demonstrable ECM, and tightly packed cells with desmosome-like junctions. In order to characterize further the ECM in the most commonly observed variant, the large light colony, specific antibodies to fibronectin (FN) and collagen types IV and V (COLs IV and V) were applied and observed with immunofluorescence microscopy and immunoperoxidase. In paraffin sections of melanoma colonies, FN was observed associated with both the cell surface and the ECM. However, no specific staining was seen for COLs IV and V. In addition, ruthenium red was used to preserve and selectively bind to glycosaminoglycans (GAGs) and proteoglycans (PGs). TEM studies reveal GAG-like granules stained with ruthenium red in the fibrillar ECM and a dotted, punctate staining of the cell surface. Understanding the biological and architectural composition of developing melanoma tumor colonies in soft agar could contribute to the development of more efficient chemotherapeutic strategies.

Topics: Agar; Cell Adhesion; Collagen; Extracellular Matrix; Fibronectins; Humans; Immunohistochemistry; Melanoma; Microscopy, Electron; Neoplasm Metastasis; Tumor Cells, Cultured

Cloning efficiency in hard agar (0.6%) and high chemotactic migration toward fibroblast conditioned medium have been shown to characterize metastatic tumor cells. We studied growth in 0.6% agar and chemotaxis of two lines of Rous Sarcoma virus-transformed Balb/c3T3 cells, B77/3T3 (low metastatic) and AA12 (high metastatic), and compared them to their non-transformed counterpart, in order to verify whether these properties were maintained during several subcultivations. Cells were cryopreserved at early passages and thawed for experiments. Both assays were performed on freshly thawed cells (4-6 weeks in culture) and on cells which had been cultured 15-20 weeks after thawing. B77/3T3, which are tumorigenic but low metastatic and which have a very low cloning efficiency in hard agar (0.1-1%), showed a chemotactic response to Balb/c3T3 conditioned medium about two-fold higher than control Balb/c3T3. This response did not change with time in culture. AA12 cells, a genetic unstable variant of B77/3T3 selected for its growth in hard agar (0.6%), had a high cloning efficiency in hard agar and showed a high chemotactic motility (three-fold the controls). High growth in 0.6% agar and high chemotaxis of AA12 were lost in late passages, where cells behaved as the controls. It seems that besides the already reported variation in anchorage-independent growth, genetically unstable tumor cells can also have important variations in chemotactic motility during subcultivations.

Topics: Agar; Animals; Avian Sarcoma Viruses; Cell Transformation, Viral; Chemotaxis; Fibroblasts; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Tumor Cells, Cultured; Tumor Stem Cell Assay

Earlier studies showed that the extracellular matrix and the conditioned medium from colon carcinoma support and catalyze the conversion of normal epithelial to colon carcinoma cells (14, 16). Since the cause of this apparent change in malignant potential is completely unknown, the following experiments examined molecular changes accompanying and mediating the transition. Colon epithelial cell cultures were initiated from normal colon biopsies. The cell cultures were carried on in standard medium on fibronectin-coated plates, and in conditioned medium on extracellular matrix from confirmed colon carcinoma. At time intervals, during 12 months period, aliquots were harvested, transferred into roller bottles to obtain enough cells to isolate cellular Poly(A)+-enriched RNA, cell membrane oligosaccharides and to determine cellular growth characteristics. During the 12 months in vitro culture, normal colon epithelial (NCE) cells grown on fibronectin in the standard growth medium maintained their initial characteristics. Whereas, NCE cells grown on the extracellular matrix and in colon carcinoma conditioned medium, progressively acquired the ability to form colonies in soft agar, to form tumors when implanted subcutaneously and to metastasize into the liver when administered intravenously into athymic mice. Poly(A)+-enriched RNA from NCE cells grown on fibronectin and in standard culture medium, did not, whereas the RNA from NCE cells grown on the extracellular matrix and in the colon carcinoma conditioned medium hybridized with 32P-cDNA from colon carcinoma. There were significant changes in the composition and profile of the oligosaccharides from membranes of NCE cells grown on the extracellular matrix. There was significant correlation (P less than 0.001) between the last characteristics.

Topics: Agar; Animals; Cell Membrane; Cells, Cultured; Colon; Colonic Neoplasms; Culture Media; Epithelial Cells; Epithelium; Female; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Oligosaccharides; Tumor Stem Cell Assay

We developed a method to determine the amount of work performed by cells through cell division in 1.0% agar cultures. There was no correlation between the cloning efficiencies of 1.0 and 0.3% agar cultures. Growth in 1.0% agar cultures correlated well with such malignant properties as tumorigenicity and the invasive and metastatic potentials. Our method revealed that metastatic MC and F cell lines possess different means of taking advantage of energy to proliferate against an environmental pressure from those possessed by nontumorigenic (ME and T-C3H) cell strain/line or nonmetastatic but tumorigenic (L,MR, and magc1) cell lines.

Topics: Agar; Animals; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Culture Media; Elasticity; Mice; Mice, Inbred A; Mice, Inbred C3H; Models, Biological; Muscles; Neoplasm Metastasis; Organ Culture Techniques; Pressure; Tumor Cells, Cultured

Spontaneous primary mammary tumors of C3H-Avy mice differ in metastatic colonization potential, some producing many lung deposits (high-colonization potential) and others producing few or none (low-colonization potential) after iv inoculation of cells. The degree of metastasis from undisturbed neoplasms also varies from tumor to tumor. This study examined whether these differences between tumors could be accounted for by differences in clonogenic or stem cell content. Tests for clonogenic cells were: growth in 0.3% agarose and limiting dilution assays. Mammary tumor cells of known colonization potential were inoculated iv at serially reduced doses, and the relationship between number of cells injected and number of lung deposits formed was determined. Parallel in vitro dose-response assays in 0.3% agarose were performed with the use of cells from the same primary tumors. Colony-forming efficiency in 0.3% agarose cultures varied between individual primary mammary tumors and was positively associated with experimental metastatic potential, suggesting that the stem or clonogenic cell content of primary tumors is one of the important determinants of the metastatic phenotype.

Topics: Agar; Animals; Colony-Forming Units Assay; Culture Media; Female; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Inbred C3H; Neoplasm Metastasis; Sepharose; Tumor Stem Cell Assay

Thirty-one bone marrow aspirations were performed on patients with prostatic carcinoma metastatic to bone. After separation over a Ficoll-Hypaque gradient viable nucleated cells were cultured in semisolid agar. Colony formation occurred in 14 of 27 (52%) nonbacterially contaminated cultures. Characterization of cells from the colonies showed them to be consistent with malignant prostate cells. After staining, these cells were periodic acid-Schiff positive, prostatic acid phosphatase positive, and prostatic specific antigen positive. Other studies demonstrated the cells to be karyotypically abnormal, ultrastructurally similar to epithelial cells, and capable of secondary colony formation. Three bone marrow aspirate specimens did not have metastatic prostatic carcinoma detected by standard methods but did demonstrate colony formation. However, colony formation was most frequently seen when a radionuclide scan was positive at the aspiration site and when tumor cells were microscopically detectable by Wright staining of a smeared aspirate. The potential utility of colony forming cultures in prostate cancer is discussed. In working with bone marrow aspirates, additional cell separation procedures may be required to calculate and maximize plating efficiencies.

Topics: Agar; Biopsy; Bone Marrow; Bone Neoplasms; Carcinoma; Cells, Cultured; Humans; Karyotyping; Male; Microscopy, Electron; Neoplasm Metastasis; Prostatic Neoplasms

Topics: Agar; Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Neoplasm Metastasis; Neoplastic Stem Cells; Rats; Tumor Stem Cell Assay

A continuous human cell line was established from a patient with large cell anaplastic lung carcinoma. This cell line, designated QU-DB, has been in culture for over 36 months and grows as an adherent monolayer with a doubling time of 10-12 hours. Its morphology, ultrastructure, karyotype, ability to grow in soft agar and heterotransplantability, indicate it is a large-cell lung tumor cell line of human origin. Three cell lines were established from metastatic tumors in nude mice receiving subcutaneous injections of QU-DB cells. The morphology and growth characteristics exhibited by these cell lines were similar to the primary cell line. Karyotypic analysis of cell lines derived from the primary tumor and a metastasis to the diaphragm were similar, but cells from a liver metastasis culture showed additional karyotypic changes. This large cell lung tumor cell line may prove useful as a model system for studies of human tumor progression and metastasis.

Topics: Agar; Aged; Animals; Carcinoma; Cell Division; Cell Line; Clone Cells; Flow Cytometry; Humans; Karyotyping; Liver Neoplasms; Lung Neoplasms; Male; Mice; Mice, Nude; Microscopy, Electron; Neoplasm Metastasis; Smoking

Topics: Agar; Cell Count; Cell Separation; Cells, Cultured; Humans; Melanoma; Neoplasm Metastasis; Neoplastic Cells, Circulating

Three distinct dissemination-related phenotypes have been distinguished among cell subpopulations of the mouse B16 melanoma: tumorigenicity, spontaneous metastasis from subcutaneous tumors, and organ colonization following intravenous injection of cells. From a progenitor clone (G3) of tumorigenic but nonmetastatic and noncolonizing (null) cells that underwent phenotypic diversification in vitro and in vivo, 4 subclones were obtained: G3.5 (culture-generated metastatic), G3.12 (tumor-generated metastatic), G3.15 (culture-generated null), and G3.26 (tumor-generated colonizing). The growth potentials of the parent clone and derived subclones were investigated comparatively in in vivo assays (tumorigenicity, tumor growth rate, and lung colonization potential), monolayer culture assays (generation time, saturation density, clonogenicity, and rate of detachment by trypsin), and in soft agar. In overall growth potential, G3.26 greater than G3.12 greater than G3, G3.5 greater than G3.15. These results indicate that metastatic populations of the B16 melanoma are not the most rapidly and effectively growing cells obtainable from that tumor.

Topics: Agar; Animals; Cell Division; Cell Movement; Cells, Cultured; Clone Cells; Female; Lung Neoplasms; Melanoma; Mice; Mice, Inbred C57BL; Microscopy, Phase-Contrast; Models, Biological; Neoplasm Metastasis; Neoplasm Transplantation; Neoplastic Stem Cells; Stem Cells

Topics: Agar; Antineoplastic Agents; Cell Division; Cells, Cultured; Humans; Neoplasm Metastasis; Neoplasms; Prognosis

Over the past year, we have attempted to grow both primary and metastatic urologic malignancies using a recently developed human tumor cloning system. Formation of colonies in vitro occurred in 125 of 164 primary tumors (76%), including 34 of 47 uroepithelial cancer specimens, 45 of 50 renal cell cancer specimens, 24 of 33 prostatic cancer specimens, and 22 of 34 testicular cancer specimens. A large percentage of metastatic cancers have also been successfully cultured. Growth sufficient for chemosensitivity testing ranged from 43% of the uroepithelial cancers cultured to 64% of the renal cell cancer specimens cultured. When in vitro chemosensitivity testing was performed, the in vitro chemosensitivity results show a striking similarity to clinical response rates for the same agents used for these tumors. Overall the human tumor cloning system appears to be a reasonable model for the study of human urologic malignancies.

Topics: Acid Phosphatase; Agar; alpha-Fetoproteins; Antineoplastic Agents; Cell Count; Chorionic Gonadotropin; Chorionic Gonadotropin, beta Subunit, Human; Clone Cells; Cytological Techniques; Drug Resistance; Genital Neoplasms, Male; Humans; Male; Microscopy, Electron; Neoplasm Metastasis; Neoplasms; Peptide Fragments; Urologic Neoplasms

Topics: Agar; Antineoplastic Agents; Cell Division; Cell Line; Cells, Cultured; Colonic Neoplasms; DNA Replication; Drug Evaluation, Preclinical; Female; Humans; Kinetics; Melanoma; Neoplasm Metastasis; Neoplasms

A procedure was developed to directly measure the self-renewal capacity of clonogenic cells from biopsies of metastatic human malignant melanoma. A culture of colony-forming cells was performed with bilayer agar in microtiter wells. The number of live tumor cells from biopsies of melanoma tissue was determined and was used to calculate plating efficiencies. Sequential photography showed that cells did not migrate in agar, thereby documenting that all the cells within colonies were direct descendants of clonogenic cells. A calibrated pneumatically controlled micropipet attached to a micromanipulator was used to quantitatively remove melanoma colonies without removing adjacent cells or agar. Plucked primary colonies were mechanically disaggregated into single cells; viability was greater than 95% as determined by trypan blue dye exclusion. Dose-related formation of secondary colonies was observed after replating of cells from pooled primary colonies. Cells from individual colonies were replated, and secondary colonies formed. These techniques allowed a simple and direct assessment of the self-renewal capacity of colony-forming melanoma cells.

Topics: Agar; Cell Division; Cells, Cultured; Clone Cells; Culture Media; Culture Techniques; Humans; Kinetics; Melanoma; Neoplasm Metastasis

Topics: Agar; Animals; Cells, Cultured; Clone Cells; Culture Media; Female; Fibrosarcoma; Mice; Neoplasm Metastasis; Neoplasms, Experimental

The influence of normal human lung fibroblasts (NLF) on the clonal growth of human prostatic carcinoma cells (PC-3) in soft agar was studied. PC-3 growth was assessed by both colony-forming efficiency and clonal growth rate as estimated from increase in colony diameter. Effects of anchored NLF (proliferating cell monolayer) and nonanchored NLF (nonproliferating cells embedded in agar) on PC-3 growth were compared. Marked differences were observed. Anchored NLF inhibited whereas nonanchored NLF stimulated PC-3. Under suboptimal growth conditions (7% fetal bovine serum), PC-3 growth was stimulated about three-fold when NLF was added to the agar at a NLF:PC-3 seeding ratio of 60:1. Similar seeding ratios using anchored NLF cells caused a 98% inhibition of PC-3 growth. Control experiments with NLF-seeded coverslips on agar cultures ruled out medium depletion as a cause of the PC-3 inhibition. PC-3 inhibition was independent of NLF cell contact, required the continued presence of NLF cells, and was thus attributed to a diffusible, labile factor derived from anchored NLF monolayers. Kinetic analysis was used to determine the nature of the nonanchored NLF-fetal bovine serum interaction on stimulated PC-3 growth. Although the NLF factor was not a component of fetal bovine serum, it acted synergistically with it to stimulate PC-3 growth. It was concluded that there were strong correlations between (a) nonanchored NLF cells and PC-3 stimulation and (b) anchored NLF cells and PC-3 inhibition.

Topics: Agar; Cell Adhesion; Cell Division; Cells, Cultured; Culture Media; Fibroblasts; Humans; Male; Neoplasm Metastasis; Prostatic Neoplasms

The pattern of in vitro anchorage-independent growth of tumor cells from the murine UV-2237 fibrosarcoma correlated with their ability to produce experimental metastasis in vivo. When seeded into 0.3% Noble agar semisolid medium, cells of metastatic clones developed into larger tumor colonies at a faster rate than did cells of clones with low metastatic potential. Furthermore, when tumor cells were plated into 0.6% Noble agar, colony development by cells of low metastatic potential clones was almost completely restricted. Tumor cells from the heterogeneous parent UV-2237 fibrosarcoma were plated into dishes containing 0.6% agar semisolid medium. In separate experiments, 16 colonies were isolated 2 weeks thereafter and were established as individual cell lines in monolayer cultures. All of these cell lines produced experimental metastases as determined by in vivo lung colony assay. The data suggest that anchorage-independent growth of UV-2237 tumor cells in 0.6% Noble agar semisolid medium is selective and permits the isolation of metastatic subpopulations.

Topics: Agar; Animals; Cell Adhesion; Cell Division; Cells, Cultured; Culture Media; Fibrosarcoma; Lung Neoplasms; Mice; Neoplasm Metastasis; Sarcoma, Experimental

Human malignant melanoma can form tumor colonies in soft agar, and at least two major morphological variants can be identified. Host cells play an important role in human melanoma colony formation, and their effects on the colony variants are selective. Neuroendocrine compounds also modulate expression of the melanoma TCFU pigmentary variants. This system appears suitable for evaluation of the response of melanoma TCFUs to chemotherapy, retinoids, heat, and radiation.

Topics: Agar; Cell Separation; Cells, Cultured; Clone Cells; Culture Media; Drug Evaluation; Growth Substances; Hormones; Humans; Lymphocytes; Melanoma; Neoplasm Metastasis; Phagocytes; Vitamin A

Fourteen breast cancer lines (8 human, 5 rat, and 1 mouse) have been studied in terms of their ability to form multicellular tumor spheroids (MTS) with the agar-base method. Only 8 of the lines formed MTS in contrast to a 100% efficiency in a series of 11 varied tumors reported in the initial studies with this method. We have compared the lines that do and do not form MTS in terms of a variety of characteristics (e.g., estrogen receptors, time in serial passage, growth in nude mice, etc.), and only one characteristic, the source of the original tumor cells, was predictive of MTS-forming ability. All 8 of the breast cancer lines (and the original 11 lines) that formed MTS had been obtained from solid growths (primaries or metastases), while the 6 breast cancer lines that did not form MTS were all derived from pleural effusions. Similarly, artificial selection for an ascites variant of the MTS-forming rat 13762 adenocarcinoma line produced the 13762-A line, which could no longer form MTS. These results suggest that breast cancer cells derived from pleural effusions are genetically different from the bulk of the tumor cells in solid breast cancer samples, that they are unable to grow in true solid form, and that these differences persist in spite of prolonged propagation in tissue culture.

Topics: Adaptation, Physiological; Agar; Animals; Breast Neoplasms; Cell Aggregation; Cell Line; Culture Techniques; Female; Humans; Mammary Neoplasms, Experimental; Mice; Neoplasm Metastasis; Neoplasms, Experimental; Pleural Effusion; Rats

Topics: Agar; Aged; Blood Protein Electrophoresis; Breast Neoplasms; Carcinoma, Bronchogenic; Colonic Neoplasms; Esophageal Neoplasms; Female; gamma-Globulins; Humans; Kidney Neoplasms; Lung Neoplasms; Middle Aged; Neoplasm Metastasis; Neoplasms; Stomach Neoplasms

Topics: Agar; Antineoplastic Agents; Female; Heparinoids; Humans; Leukemia; Male; Neoplasm Metastasis; Neoplasms; Radiation Effects